Genome-wide localization of histone variants in Toxoplasma gondii implicates variant exchange in stage-specific gene expression.

BACKGROUND: Toxoplasma gondii is a protozoan parasite that differentiates from acute tachyzoite stages to latent bradyzoite forms in response to environmental cues that modify the epigenome. We studied the distribution of the histone variants CenH3, H3.3, H2A.X, H2A.Z and H2B.Z, by genome-wide chromatin immunoprecipitation to understand the role of variant histones in developmental transitions of T. gondii parasites. RESULTS: H3.3 and H2A.X were detected in telomere and telomere associated sequences, whereas H3.3, H2A.X and CenH3 were enriched in centromeres. Histones H2A.Z and H2B.Z colocalize with the transcriptional activation mark H3K4me3 in promoter regions surrounding the nucleosome-free region upstream of the transcription start site. The H2B.Z/H2A.Z histone pair also localizes to the gene bodies of genes that are silent but poised for activation, including bradyzoite stage-specific genes. The majority of H2A.X and H2A.Z/H2B.Z loci do not overlap, consistent with variant histones demarcating specific functional regions of chromatin. The extent of enrichment of H2A.Z/H2B.Z (and H3.3 and H2A.X) within the entire gene (5'UTR and gene body) reflects the timing of gene expression during the cell cycle, suggesting that dynamic turnover of H2B.Z/H2A.Z occurs during the tachyzoite cell cycle. Thus, the distribution of the variant histone H2A.Z/H2B.Z dimer defines active and developmentally silenced regions of the T. gondii epigenome including genes that are poised for expression. CONCLUSIONS: Histone variants mark functional regions of parasite genomes with the dynamic placement of the H2A.Z/H2B.Z dimer implicated as an evolutionarily conserved regulator of parasite and eukaryotic differentiation

Essential role of Plasmodium perforin-like protein 4 in ookinete midgut passage.

Pore forming proteins such as those belonging to the membrane attack/perforin (MACPF) family have important functions in many organisms. Of the five MACPF proteins found in Plasmodium parasites, three have functions in cell passage and one in host cell egress. Here we report an analysis of the perforin-like protein 4, PPLP4, in the rodent parasite Plasmodium berghei. We found that the protein is expressed only in the ookinete, the invasive stage of the parasite formed in the mosquito midgut. Transcriptional analysis revealed that expression of the pplp4 gene commences during ookinete development. The protein was detected in retorts and mature ookinetes. Using two antibodies, the protein was found localized in a dotted pattern, and 3-D SIM super-resolution microcopy revealed the protein in the periphery of the cell. Analysis of a C-terminal mCherry fusion of the protein however showed mainly cytoplasmic label. A pplp4 null mutant formed motile ookinetes, but these were unable to invade and traverse the midgut epithelium resulting in severely impaired oocyst formation and no transmission to naïve mice. However, when in vitro cultured ookinetes were injected into the thorax of the mosquito, thus by-passing midgut passage, sporozoites were formed and the mutant parasites were able to infect naïve mice. Taken together, our data show that PPLP4 is required only for ookinete invasion of the mosquito midgut. Thus PPLP4 has a similar role to the previously studied PPLP3 and PPLP5, raising the question why three proteins with MACPF domains are needed for invasion by the ookinete of the mosquito midgut epithelium

Molecular epidemiology and expression of capsular polysaccharides in Staphylococcus aureus clinical isolates in the United States.

Staphylococcus aureus capsular polysaccharides (CP) are important virulence factors under evaluation as vaccine antigens. Clinical S. aureus isolates have the biosynthetic capability to express either CP5 or CP8 and an understanding of the relationship between CP genotype/phenotype and S. aureus epidemiology is valuable. Using whole genome sequencing, the clonal relatedness and CP genotype were evaluated for disease-associated S. aureus isolates selected from the Tigecycline Evaluation and Surveillance Trial (T.E.S.T) to represent different geographic regions in the United States (US) during 2004 and 2009-10. Thirteen prominent clonal complexes (CC) were identified, with CC5, 8, 30 and 45 representing >80% of disease isolates. CC5 and CC8 isolates were CP type 5 and, CC30 and CC45 isolates were CP type 8. Representative isolates from prevalent CC were susceptible to in vitro opsonophagocytic killing elicited by anti-CP antibodies, demonstrating that susceptibility to opsonic killing is not linked to the genetic lineage. However, as not all S. aureus isolates may express CP, isolates representing the diversity of disease isolates were assessed for CP production. While approximately 35% of isolates (primarily CC8) did not express CP in vitro, CP expression could be clearly demonstrated in vivo for 77% of a subset of these isolates (n = 20) despite the presence of mutations within the capsule operon. CP expression in vivo was also confirmed indirectly by measuring an increase in CP specific antibodies in mice infected with CP5 or CP8 isolates. Detection of antigen expression in vivo in relevant disease states is important to support the inclusion of these antigens in vaccines. Our findings confirm the validity of CP as vaccine targets and the potential of CP-based vaccines to contribute to S. aureus disease prevention

Potential for maternally administered vaccine for infant group B streptococcus

BACKGROUND : Natural history studies have correlated serotype-specific anti–capsular polysaccharide (CPS) IgG in newborns with a reduced risk of group B streptococcal disease. A hexavalent CPS–cross-reactive material 197 glycoconjugate vaccine (GBS6) is being developed as a maternal vaccine to prevent invasive group B streptococcus in young infants. METHODS : In an ongoing phase 2, placebo-controlled trial involving pregnant women, we assessed the safety and immunogenicity of a single dose of various GBS6 formulations and analyzed maternally transferred anti-CPS antibodies. In a parallel seroepidemiologic study that was conducted in the same population, we assessed serotype-specific anti-CPS IgG concentrations that were associated with a reduced risk of invasive disease among newborns through 89 days of age to define putative protective thresholds. RESULTS : Naturally acquired anti-CPS IgG concentrations were associated with a reduced risk of disease among infants in the seroepidemiologic study. IgG thresholds that were determined to be associated with 75 to 95% reductions in the risk of disease were 0.184 to 0.827 μg per milliliter. No GBS6-associated safety signals were observed among the mothers or infants. The incidence of adverse events and of serious adverse events were similar across the trial groups for both mothers and infants; more local reactions were observed in the groups that received GBS6 containing aluminum phosphate. Among the infants, the most common serious adverse events were minor congenital anomalies (umbilical hernia and congenital dermal melanocytosis). GBS6 induced maternal antibody responses to all serotypes, with maternal-to-infant antibody ratios of approximately 0.4 to 1.3, depending on the dose. The percentage of infants with anti-CPS IgG concentrations above 0.184 μg per milliliter varied according to serotype and formulation, with 57 to 97% of the infants having a seroresponse to the most immunogenic formulation. CONCLUSIONS : GBS6 elicited anti-CPS antibodies against group B streptococcus in pregnant women that were transferred to infants at levels associated with a reduced risk of invasive group B streptococcal disease.Pfizer and the Bill and Melinda Gates Foundation.http://www.nejm.orghj2024Medical MicrobiologySDG-03:Good heatlh and well-bein

Global identification of multiple substrates for Plasmodium falciparum SUB1, an essential malarial processing protease.

The protozoan pathogen responsible for the most severe form of human malaria, Plasmodium falciparum, replicates asexually in erythrocytes within a membrane-bound parasitophorous vacuole (PV). Following each round of intracellular growth, the PV membrane (PVM) and host cell membrane rupture to release infectious merozoites in a protease-dependent process called egress. Previous work has shown that, just prior to egress, an essential, subtilisin-like parasite protease called PfSUB1 is discharged into the PV lumen, where it directly cleaves a number of important merozoite surface and PV proteins. These include the essential merozoite surface protein complex MSP1/6/7 and members of a family of papain-like putative proteases called SERA (serine-rich antigen) that are implicated in egress. To determine whether PfSUB1 has additional, previously unrecognized substrates, we have performed a bioinformatic and proteomic analysis of the entire late asexual blood stage proteome of the parasite. Our results demonstrate that PfSUB1 is responsible for the proteolytic processing of a range of merozoite, PV, and PVM proteins, including the rhoptry protein RAP1 (rhoptry-associated protein 1) and the merozoite surface protein MSRP2 (MSP7-related protein-2). Our findings imply multiple roles for PfSUB1 in the parasite life cycle, further supporting the case for considering the protease as a potential new antimalarial drug target

Correction: Essential role of Plasmodium perforin-like protein 4 in ookinete midgut passage.

[This corrects the article DOI: 10.1371/journal.pone.0201651.]

Molecular epidemiology and expression of capsular polysaccharides in <i>Staphylococcus aureus </i>clinical isolates in the United States

Staphylococcus aureus capsular polysaccharides (CP) are important virulence factors under evaluation as vaccine antigens. Clinical S. aureus isolates have the biosynthetic capability to express either CP5 or CP8 and an understanding of the relationship between CP genotype/phenotype and S. aureus epidemiology is valuable. Using whole genome sequencing, the clonal relatedness and CP genotype were evaluated for disease-associated S. aureus isolates selected from the Tigecycline Evaluation and Surveillance Trial (T.E.S.T) to represent different geographic regions in the United States (US) during 2004 and 2009–10. Thirteen prominent clonal complexes (CC) were identified, with CC5, 8, 30 and 45 representing &gt;80% of disease isolates. CC5 and CC8 isolates were CP type 5 and, CC30 and CC45 isolates were CP type 8. Representative isolates from prevalent CC were susceptible to in vitro opsonophagocytic killing elicited by anti-CP antibodies, demonstrating that susceptibility to opsonic killing is not linked to the genetic lineage. However, as not all S. aureus isolates may express CP, isolates representing the diversity of disease isolates were assessed for CP production. While approximately 35% of isolates (primarily CC8) did not express CP in vitro, CP expression could be clearly demonstrated in vivo for 77% of a subset of these isolates (n = 20) despite the presence of mutations within the capsule operon. CP expression in vivo was also confirmed indirectly by measuring an increase in CP specific antibodies in mice infected with CP5 or CP8 isolates. Detection of antigen expression in vivo in relevant disease states is important to support the inclusion of these antigens in vaccines. Our findings confirm the validity of CP as vaccine targets and the potential of CP-based vaccines to contribute to S. aureus disease prevention